The present invention relates to the fields of cellular biology and internal medicine and to a method of treating or preventing a respiratory distress syndrome and, more particularly, to the activation and regulation of surfactant secretion in type II alveolar pneumocytes.
A physiologically active substance, called xe2x80x9cpulmonary surfactantxe2x80x9d exists in the animal lungs. Pulmonary surfactant is mainly biosynthesized in and secreted from type II epithelial cells of the alveoli and is known to be present as an internal lining of the wall of t he whole respiratory tract including the alveolar region. It is known that pulmonary surfactant reduces the surface tension of the alveoli and prevents collapse of the alveoli. The ability of surfactant to reduce surface tension means that less effort is needed to re-inflate the lungs after the alveoli are drained of air during exhalation. The less effort required, the less trauma to the lung itself in the course of normal breathing.
In addition, pulmonary surfactant plays an important role as a defense mechanism in the entire respiratory tract. It is well documented that it prevents pulmonary edema and has preventative effects on bacterial infection, viral infection, as well as on atmospheric pollutants and antigens which induce inflammation of the respiratory tract or asthmatic attacks. Pulmonary surfactant is also known to play an important role in lubricating the respiratory lumen and expelling foreign matter from the respiratory tract by activating mucociliary transport.
Pulmonary surfactant is a complex mixture of proteins and phospholipids. There are four known proteins in alveolar surfactant; SP-A, -B, -C, and -D. SP-B and -C are small, very hydrophobic proteins that interact with phospholipids to lower alveolar surface tension. SP-D is a 43 kDa apoprotein of uncertain function. Like SP-A, SP-D has collagen-like domains. SP-A is a moderately hydrophobic 29-36 kDa apo-protein. It reportedly stabilizes the phospholipid structure and promotes interactions between phospholipids. It also appears to be important in regulating surfactant secretion. These proteins, together with phospholipids, are secreted from alveolar type II pneumocytes and form the air-liquid interphase in the alveoli and comprise what is referred to herein as xe2x80x9calveolar surfactantxe2x80x9d.
Because of its various physiological functions in the respiratory system, qualitative and quantitative changes of pulmonary surfactant seem to be related to the onset of or aggravation of many conditions. Accordingly, the modulation of secretion of pulmonary surfactant will allow for the treatment or prevention of various respiratory conditions, including, but not limited to, acute respiratory failure such as infant or adult respiratory distress syndrome, bronchitis, infectious disease and chronic respiratory failure.
The mechanism(s) that activate and regulate surfactant secretion are not well understood, but evidence suggests that calcium is important in signaling this process. The role of calcium signaling in the activation of surfactant secretion reflects changes in the concentration of free calcium in cytosol stores (lumenal calcium concentration, or [Ca2+]l) and is independent of the cytosolic calcium concentration ([Ca2+]i).
Surfactant secretion by type II cells is often studied in vitro, using receptor-binding secretagogues such as purines or xcex1-adrenergic agonists. (Sano, et al, Am. J. Physiol. 253: C679-C686, 1987; Sen, et al, Biochem. J. 298:681-687, 1994; Strayer, et al, Exp. Cell Res. 226: 90-97, 1996; Strayer, et al, Rec. Signal Transd. 7: 111-120, 1997). Surfactant secretion may also be activated independently of cell membrane receptors. Calcium ionophores (such as ionomycin (Io), are surfactant secretagogues that lack plasma membrane receptors. They release calcium from ER stores directly, and carry calcium from outside the cell into the cytosol. The secretagogue activity for thapsigargin (TG) also stimulates surfactant secretion. (Strayer, et al, Rec. Signal Transd. 7: 111-120, 1997; Thastrop, et al, Proc. Natl. Acad. Sci. USA 87: 2466-2470, 1990). Thapsigargin directly inhibits the Ca2+-dependent ATPase pump that maintains the calcium gradient at the ER stores, thereby acting directly on stores to increase [Ca2+]i. These secretagogues emphasize the importance of ER calcium stores in signaling surfactant secretion (Strayer, et al, Exp. Cell Res. 226: 90-97, 1996). With or without receptor mediation, these secretagogues elicit rapid, large changes in [Ca2+]i. Calcium is released from intracellular stores, followed by influx of calcium through plasma membrane calcium channels. (Strayer, et al, Rec. Signal Transd. 7: 111-120, 1997; Berridge, M J, Nature, 361: 315-325, 1993). A potent surfactant secretagogue that is an exception are the phorbal esters. They stimulate surfactant secretion without altering [Ca2+]i. (Sano, et al, Am. J. Physiol. 253: C679-C686, 1987).
One of the surfactant proteins, SP-A, decreases secretagogue-stimulated surfactant secretion. (Strayer, et al, Exp. Cell Res 222: 681-687, 1994; Dobbs, et al, Proc. Natl. Acad. Sci. USA 84:1010-1014, 1987; Hawgood and Shiffer, Annu. Rev. Physiol. 53: 375-394, 1991; Rice, et al, J. Appl. Physiol. 63: 692-698, 1987 Rooney, et al, FASEB J. 8, 957-967, 1994). SP-A binds a type II cell membrane receptor (SPAR) to prevent the Ca2+ release elicited by all the above secretagogues, including those acting directly on stores (Io and TG). (Strayer, et al, Rec. Signal Transd. 7: 111-120, 1997). SP-A does not block transmembrane Ca2+ fluxes, whether active (i.e., via voltage or other gated channels) or passive (e.g., via Io). (Strayer, et al, Rec. Signal Transd. 7: 111-120, 1997).
Signaling Mechanisms in Surfactant Secretion
The signaling mechanisms that stimulate and inhibit surfactant secretion are not well understood. The diversity of secretagogues, some with different receptors, some without receptors, each with separate signaling pathways, greatly complicates the task of elucidating how surfactant secretion is triggered. (Chander and Fisher, Am. J. Physiol. 258: L241-253, 1990; Rotonda, et al, Thromb. Haemost. 78: 919-925, 1997). The nature of the signal that begins at the SP-A receptor (SPAR) and down-regulates surfactant secretion is even more obscure.
Adenosine binds A2 purine receptors to activate adenylate cyclase via G protein-dependent pathways to produce cAMP. cAMP activates protein kinase A. Alpha-adrenergic agonists act similarly, though through different receptors. ATP binds P2y and P2u receptors to signal via G proteins to activate phospholipase Cxcex2 (phosphoinositide-specific phospholipase C, PLCxcex2). PLCxcex2 hydrolyzes phosphatidylinositol-3,4,5-trisphosphate to diacyl glycerol (DAG) and inositol-3,4,5-trisphosphate, both of which activate other enzymes, such as protein kinases C, phospholipase D, etc. Secretagogue-induced signaling in type II cells downstream from these points is poorly understood.
Intracellular calcium stores in different cell types possess several types of receptors that can be stimulated to cause Ca2+ release. (Berridge, M J. Nature 361:315-325, 1993; Mikoshiba, K., Curr. Opin. Neurobiol. 7:339-345, 1997). Ryanodine and IP3 receptors (IP3R) are examples of proteins that traverse the ER membrane and release Ca2+ on binding their ligands. Calcium release (or increased [Ca2+]i) may activate tyrosine-specific protein kinases and calmodulin-dependent kinases. (Sugden, et al, Cell. Signal. 9:337-351, 1997). Again, however, the means by which this signaling mechanism would result in surfactant secretion is unclear.
Downstream signaling that follows calcium release from stores is an area of intense investigation. In type II cells, calcium release activates cell membrane calcium channels, allowing influx of Ca2+. (Berridge, M J, Nature 361:315-325, 1993; Putney, J W, Jr., Science, 262: 676-678, 1993; Putney, J W, Jr., Cell, 75, 199-201, 1993). The nature of the stimulus/stimuli that opens Ca2+ channels is under debate. Candidates range from increased [Ca2+]i to poorly characterized substances that may be released simultaneously with Ca2+ from those stores. (Berridge, M J, Nature 361:315-325, 1993).
Surfactant Deficiency
Surfactant is essential for normal respiratory function. Diseases of surfactant deficiency are characterized by respiratory distress. Thus, when insufficient surfactant is present to lower surface tension, a great deal of energy is needed to reinflate alveoli. Consequently, inhalation causes damage to the alveolar lining cells (mainly type I pneumocytes). Surfactant insufficiency, whether due to prematurity, inactivation or genetic deficiency, is not compatible with extrauterine life. (Avery, et al, AM. J. Dis. Child. 97: 517-523. 1959; Lewis, et al., Am. Rev. Resp. Dis. 147: 218-233, 1993; Nogee, et al, J. Clin. Invest. 93: 1860-1863, 1994).
Pulmonary immaturity, termed neonatal respiratory distress syndrome (RDS) is a disease that occurs in infants born prematurely. It occurs in about 10,000 infants yearly in the U.S. In many such infants, their type II cell surfactant secretory apparatus has not matured adequately to sustain the levels of surfactant secretion needed for extrauterine life. The level of surfactant production, i.e., the level of pulmonary maturity at which extrauterine life can be sustained, has decreased greatly in recent years with improved ventilatory technology. The development of exogenous surfactant replacement therapy in such infants has provided an additional type of therapeutic intervention that has decreased both morbidity and mortality of neonatal RDS greatly. Currently, with surfactant replacement therapy and modem ventilatory techniques the mortality from neonatal RDS in infants who are born 26-28 weeks is less than 20%, and in some centers as low as 10%.
Unfortunately, surfactant replacement therapy is expensive. One dose for a 750 to 1000 gram infant costs about $1200, although the individual cost varies with the specific formulation. Many infants require more than one dose. Further, surfactant replacement therapy fails for unknown reasons in about 20% of premature infants in whom it is used.
Surfactant deficiency that is acquired may occur at any time in life, but generally involves adults. Statistics on its frequency are difficult to obtain because these patients are often classified according to initial extrapulmonary diseases (e.g., automobile accidents). Still, about 50,000 adult respiratory deficiency syndrome (ARDS) cases occur in the U.S. yearly.
This deficiency is a relative deficiency. Type II cells are mature, and are generally metabolically active and capable of producing and secreting surfactant. In ARDS, however, the alveolar space may contain inhibitors of surfactant function that, even in the presence of normal concentrations of surfactant, impede the surface activity of the lung""s secreted surfactant. The surfactant inhibitors are usually considered to be natural plasma proteins that do not normally reach the air spaces of the lung, for example fibrinogen and albumin. Occasionally, in the setting of infection, microbial inhibitors of surfactant may also be involved.
Adult respiratory distress syndrome (ARDS) is a complex syndrome often involving substantial extrapulmonary disease. Pulmonary disease commonly represents one of a number of major organ system failures. ARDS may occur in the setting including, but not limited to, sepsis (i.e., overwhelming bacterial infection), traumatic shock (i.e., insufficient blood volume to maintain normal organ function, as can occur after an automobile accident), cardiogenic shock (heart failure), toxin exposure either by inhalation or other routes, and numerous other insults including fresh water drowning, severe radiation exposure, pneumonia, etc.
ARDS does not occur in all people exposed to these injuries. The reasons ARDS occurs in some and not others is unclear. The mortality of ARDS in the U.S. is about 50-60%, and has not changed greatly since the syndrome was first recognized in the early 1970s. Patients often die from their extrapulmonary diseases (e.g., overwhelming infection).
Treatment for ARDS is not very effective, and has not improved mortality greatly despite improved ventilatory therapy. This reflects in part the general complexity of these patients"" clinical situations and in part the unavailability of surfactant replacement for ARDS patients. As indicated above, one dose of, for example, bovine surfactant for a 750 gram infant costs over $1000. A 75 kilogram adult would need at least 100xc3x97 as much, and may well require several doses. While anecdotal reports in a small series of patients suggest that surfactant administration is helpful in ARDS, both availability and cost are major concerns.
To date, genetic deficiency of one of the surfactant proteins, surfactant protein B (SP-13) has been described in a small but growing population of infants. This disease is invariably fatal unless a lung transplant is performed in the neonatal period.
Given the high cost and lack of adequate therapies in the current protocols for treating a respiratory distress syndrome, there is a long felt, yet unfulfilled need, for additional therapies for respiratory distress. The present invention fulfills this need. The present invention examines the role of Ca2+ in signaling surfactant secretion. The results imply that a novel role for calcium stores in cellular activation exists in type II alveolar pneumocytes.
BAPTA-AM, an esterified EGTA analogue, does not bind Ca2+ and crosses cell membranes freely. It is de-esterified in the cytosol, whereupon it binds Ca2+. BAPTA-AM causes a huge increase in surfactant secretion. It is non-toxic and commonly available. The present invention relates to the chelation of intracellular calcium, with for example BAPTA-AM, and to the stimulation of surfactant secretion by type II cells. The present invention is a major advance in pulmonary therapeutics. For a tiny fraction ( less than 1%) of the cost of administering exogenous surfactant to a patient, the calcium chelator, for example BAPTA-AM, is instilled via a number of potential delivery mechanisms and will cause type II cells to release stored surfactant pools. Thus, the present invention provides a therapeutic/prophylactic method of treating a patient with a calcium chelator, thereby stimulating surfactant secretion by type II pneumocytes and facilitating a normal breathing pattern.
An object of the invention is to present a method of treating or preventing a respiratory distress syndrome in a mammal by administering a therapeutically effect amount of an agent that activates surfactant secretion in the mammal.
It is a further object of the present invention that the therapeutic agent used in the method of treatment or prevention of a respiratory distress syndrome uses at least one intracellular calcium chelator. In one embodiment of the invention the intracellular calcium chelator is BAPTA-AM. The BAPTA-AM is between 25 and 100 xcexcM.
It is another object of the present invention that the therapeutic agent used in the method of treatment or prevention of a respiratory distress syndrome enhances secretion of surfactant from type II pneumocytes. In one embodiment the therapeutic agent acts by altering the endoplasmic reticulum free calcium concentration ([Ca+2]l] in the type II pneumocytes.
It is also an object of the present invention that the therapeutic agent is administered by aerosol, nebulization or liquid instillation.